This proposal is a new submission for a FIRST award to study the regulation of expression of beta-lactamase and penicillin-binding protein 2a (PBP2a), which are responsible for resistance to the beta-lactam class of antibiotics in Staphylococcus aureus. S. aureus is a common cause of nosocomial as well as community-acquired infections in the United States. Resistance to penicillin mediated by plasmid-encoded beta-lactamase developed rapidly and now occurs in over 95% of strains. Broader resistance to methicillin and all other beta-lactam antibiotics is mediated by PBP2a and is prevalent in many hospitals. Expression of beta-lactamase, which degrades penicillin, and PBP2a, a cell wall synthesis enzyme with low affinity for beta-lactams, is inducible upon exposure to beta-lactams, and regulation involves a putative repressor protein (BlaI) and a signal-transducing membrane protein (BlaR1). BlaR1 has an extracellular domain that binds beta-lactam antibiotics and by an unknown mechanism signals the cell to produce the resistance factors. The long-term objective of the proposal is to dissect and understand this signal-transducing mechanism that regulates beta-lactamase and PBP2a production. Determining the mechanisms involved in the induction of these two proteins will increase the understanding of how S. aureus develops resistance to beta-lactams. The specific aims of the proposal are: 1) characterize the properties of the beta-lactamase repressor protein, BlaI, by measuring its interaction with the putative operator region of blaZ, which encodes beta-lactamase, and performing site-directed mutagenesis on blaI; 2) characterize the functional roles of the extracellular and cytoplasmic domains of the signal transducer, BlaR1, by correlating the affinity of beta-lactams for BlaR1 with their potency as inducers and studying the interaction of the BlaR1 cytoplasmic domain with BlaI and other cellular components; and 3) analyze by genetic techniques the S. aureus beta-lactamase regulatory system using construction of uninducible and constitutive mutants.